Two Quenchers Are Better Than One!

Including a second, internal quencher in qPCR probes shortens the distance between 5’ dye and quencher and, in concert with the 3’ quencher, provides greater overall dye quenching, lowering background and increasing signal detection in qPCR experiments (Figure 1A). The decrease in background enables the use of more probes in a multiplex qPCR experiment by reducing crosstalk between channels; additionally, multiple probes can be used to test for the presence of a gene in one detector channel (see Figure 1 in the article, Optimizing Multiplex qPCR for Detecting Infectious Disease and Biothreat Agents in the Field). The additional ZEN™ quencher also increases assay sensitivity, as evidenced by earlier Cq values (Figure 1B). This greater sensitivity can be crucial for detecting targets that are limited due to small sample size or low target expression. In fact, the performance of ZEN Double-Quenched Probes has enabled applications that would otherwise not have been possible [1].

Figure 2 shows that the decrease in background fluorescence is also evident with longer probes. The ability to use longer probes without sacrificing assay performance provides greater flexibility when designing assays; e.g., when interrogating regions of low complexity, it may be necessary to design longer probes to achieve a higher Tm. Use of ZEN Double-Quenched Probes provides researchers both increased sensitivity and precision in their qPCR experiments. qPCR probes that include the ZEN quencher (Double-Quenched Probes) can be ordered online at www.idtdna.com (look for Prime- Time® qPCR Probes).